This R21 pilot study will demonstrate improved methods for whole cell antibacterial activity screening and biochemical target identification. This effort will be driven by two key innovations. First will be the use of pre-metabolized (pre-met) compound libraries. Pre-met will increase library chemical diversity in a human biology relevant dimension, and in a way well-aligned with known antibacterial agent characteristics, and it is hypothesized that this increased diversity will give significantly higher (>3xfold) hit rates than n-metabolized libraries. This approach will also allow the early identification of toxic or otherwise unfavorable compounds, further improving library screening efficiency. The second key innovation will be the use of high information content LC-MS/MS-based peptidogly can metabolite quantification to rapid identify peptidoglycan biosynthesis inhibitors, using methods established and validated in preliminary studies. This effort is comprised of the following specifi aims. Specific Aim 1: Evaluate library pre-metabolization for whole cell antibacterial screening. A small compound library (4000 samples), and an FDA approved drug library (853 samples), will be screened for activity against E. coli, P. aeruginosa, S.aureus (MRSA), and E. faecium. A parallel screen using premetabolized libraries will also be performed. Hits will be characterized for MICs and MBC, and tested for hepatotoxicity. The FDA library will provide both a useful control group, and identify previously unknown antibacterial metabolites of FDA approved drugs. Specific Aim 2: Evaluate high information content assays for the rapid identification of agents targeting bacterial PG biosynthesis. Bactericidal parent agents and metabolites (purified) will be screening for PG biosynthesis targeting agents using high information content LC-MS/MS-based PG metabolite assays. These assays will also identify or localize the targeted step. The FDA approved drug library will provide a control group to demonstrate this potential. This pilot study will quantitatively evaluate the potential of the above?cited innovations to improve antibacterial agent discovery from library screening. Demonstration of pre?metabolized libraries as a means of increasing library diversity and hit rate, and as a means to eliminate hepatotoxic and other unfavorable agents from a library screen, will be of general interest and utility for othr library screening efforts. The demonstration of high content metabolite assays to quickly identify agents targeting a specific biochemical pathway can be extended too many other bacterial biochemical pathways, and is also expected to be of high general interest. This study will provide a foundation for larger scale efforts and follow-on studies to identify and develop novel antibacterial agents.